Charakterisierung von Trix, einem neuen Guaninnukleotid-Austauschfaktor aus Dictyostelium discoideum

The goal of this project was the characterisation of a new guanine
nucleotide exchange factor (GEF protein) for Rac GTPases in
Dictyostelium discoideum. GEF proteins function as stimulating
proteins of Rac GTPases which are of central importance in the
regulation of actin-involving processes. Actin is a major component
of the cytoskeleton, constituting a dynamic network of filamentous
structures which provide the basis for elementary tasks of growth,
differentiation, cell movement and cell division. The ameba D.
discoideum is a haploid unicellular eukaryote. It serves as a model
organism to study basic actin-involving processes in higher
organisms. The D. discoideum genome has been fully sequenced
recently and comprises a wide range of regulatory components for
the actin cytoskeleton. The gene of a novel GEF protein with a
coding sequence of 3597 bp was studied in detail. The gene was
cloned and the sequence verified. The protein sequence of 1198
amino acids comprises three Calponin homology domains (CH) and one
Dbl homology/Pleckstrin homology tandem domain which is a typical
sequence feature of GEF proteins. On the basis of this domain
architecture the protein was named 'Trix' in short for 'triple
Calponin exchange factor'. The CH domains were classified as type 1
and type 1 CH domain (type 3 - type 3 - type 1). This resembles a
novel combination of CH domains in a Rho GEF protein. A recombinant
fragment carrying the three CH domains was shown to bind and bundle
actin filaments which is not explained by the functions that have
been described for CH domains so far. In vivo Trix is localised in
the area of the actin-rich cell cortex as well as on the membranes
of late endocytic vesicles. This suggests a regulatory role for
Trix in the assembly or the disassembly of the actin coat that is
associated with endocytic vesicles during the late stages of
endocytosis. It was shown that Trix is mainly expressed during the
vegetative stadium of D. discoideum which would be consistent with
the increased endocytosis during growth. An association of Rac
GTPases with late endocytic vesicles in D. discoideum was not
described so far. Trix displayed no GDP/MANT-GDP exchange activity
with the Rac GTPases Rac1a, RacC and RacE, hence the protein could
not be allocated to a Rac GTPase signaling pathway. Some of the
interactions between exchange factors and their respective Rac
GTPases are of a very specific nature. Thus a potential activity of
Trix might be directed against a Rac GTPase that has not yet been
tested. The activation of Trix might also depend on further
uncharacterised regulatory components. Finally, the results of the
in vitro assays might differ from the in vivo situation as it has
been demonstrated for other Rho GEFs. The Trix gene was disrupted
in AX2 wildtype cells by a gene replacement approach. This allowed
detailed characterisation of the protein's function in vivo. Trix
is not an essential protein. There were no significant differences
in the expression of important marker proteins, in phototaxis,
chemotaxis, phagocytosis, cytokinesis, and growth of Trix- mutants.
The mutant cells showed subtle changes in the organisation of the
actin system as well as a slight delay during the developmental
cycle. The most severe phenotypic deviation displayed by Trix-
mutants consisted in a marked reduction of exocytosis. This
provides further evidence for a regulatory function of the protein
in exocytosis. The data suggest that Trix plays a role in the
organisation of actin-involving processes in D. discoideum,
especially in the regulation of the late endocytic cycle. Trix
could not be allocated to a specific Rac GTPase signaling pathway
and definite structural or dynamic tasks on the basis of the
protein’s actin-binding and actin-bundling properties. The subtle
phenotypic alterations in Trix- mutants might be due to a general
redundancy in the functions of D. discoideum Rho GEF proteins.